Multiple pathways for metabolism of estrone and estradiol are known but do not account for total metabolism of these steroids. Significant amounts are converted to 2-hydroxy estrogens, which are then transformed to unknown compounds. This project will address questions on the identity and subsequent fate of metabolites of these catechol estrogens, location of the conversion, and physiological significance of the process. Based on our previous experience with mushroom tyrosinase, we suggest that catechol estrogens are oxidized in mammalian organisms by a tyrosinase present in melanocytes and that the products undergo addition reactions to form dimers and polymers which may be stored in the tissues. 2-Hydroxyestradiol (2-OH E2) was shown to be oxidized stoichiometrically by oxygen with mushroom tyrosinase in presence of catechol as cofactor. The products appear as unidentified dark, melanin-like compounds. These products have not been described before and we intend to study their structure closely because of their possible significance as physiologically active estrogen derivatives or as estrogen catabolites which may occur in mammalian systems. We will isolate these products and subject them to ESR analysis. We will determine the mechanism of reaction by ESR techniques because free radicals are likely to be formed during or after enzyme reaction. We want to compare the fungal enzyme with mammalian tyrosinase, to be isolated from murine malignant melanomas, to determine if it, too, can metabolize 2-OH E2. Since melanomas are known to give a positive growth response to estrogens and to possess estrogen receptors, we hypothesize that a melanin-like compound may be formed from products of estrogen oxidation in melanomas. We will examine the role of mouse melanomas in 2-OH E2 metabolism by injecting 3H-2-OH E2 and determining radioactivity in isolated products and by measuring affinity of 2-OH E2 to melanocyte binding sites. This project will seek to elucidate the metabolism of catechol estrogens and their role in malignant melanoma formation.